OP0008 A NOVEL SITE-SPECIFIC PEGYLATED IL-2 WITH POTENT AND TREG-SELECTIVE ACTIVITY IN VIVO
Authors: M. Ikeda, S. Yamaguchi, M. Murakami, S. Takaoka, Y. Sakaguchi, S. Yasui, K. Iijima, K. Nanya, H. Onodera, T. Amano
Journal: Annals of the Rheumatic Diseases (2022)

Background Decreased regulatory T cells (Tregs) and Treg dysfunction are hallmarks of a various autoimmune and inflammatory diseases. While low-dose IL-2 therapy induces Treg expansion in vivo and has clinical benefits in some diseases (e.g., SLE and chronic graft-versus-host disease [GvHD]), there are many concerns about adverse events due to low Treg-selectivity. Furthermore, frequent dosing is needed due to the short half-life. Objectives We discovered a novel site-specific PEGylated IL-2 variant, KKC80, with high Treg selectivity and a long half-life in vivo , which overcomes the issues of low-dose IL-2 therapy. Methods Based on the co-crystal structure of wild-type IL-2 and its heterotrimeric receptor (PBD ID: 2ERJ), amino acid residues that were to be PEGylation sites were substituted with oAzZLys, an azide-containing lysine derivative. The PEG molecule was site-specifically attached to oAzZLys-incorporated IL-2 by copper-free click chemistry. The binding property to the IL-2 receptors were measured by surface plasmon resonance (SPR). In vitro , Treg selectivity was evaluated by the IL-2-dependent proliferation activity of Tregs and NK cells from human peripheral blood mononuclear cells (PBMCs). In vivo pharmacological activity after the single subcutaneous administration in cynomolgus monkeys was measured by changes in Treg count and Treg activation status in peripheral blood by flow cytometry. Pharmacokinetic parameters were calculated according to serum PEGylated IL-2 concentration. Efficacy in mouse xenogeneic GvHD model using human PBMC-transplanted NOG mice and in monkey DTH model were evaluated. Results A novel PEGylated IL-2, KKC80 (human IL-2 desA1/C125S /I129oAzZLys_W-shaped 80 kDa PEG) was discovered by optimizing the PEGylation site and PEG structure based on Treg selectivity and PK. SPR analysis showed that the binding affinity of KKC80 to CD25 was moderately decreased from wild-type IL-2, while binding affinity of KKC80 to IL-2Rβγ was remarkably decreased due to a significant change of the association rate constant. In vitro , wild-type IL-2 activated both Tregs and NK cells in the same concentration range, whereas KKC80 selectively activated Tregs. The Treg selectivity of KKC80 was comparable to another IL-2 mutein, Fc.IL-2 V91K. KKC80, but not Fc.IL-2 V91K, retained its biological activity, even in the presence of a large amount of recombinant soluble CD25, which mimicked the endogenous decoy receptor for IL-2. In monkeys, KKC80 selectively increased peripheral blood Tregs in a dose-dependent manner; the average maximum rate of increase of Treg count in animals treated with 0.01, 0.03, 0.1, 0.3 and 1 mg/kg was 1.5, 3.5, 28, 50 and 154-fold, respectively. In contrast to Tregs, the rates of increase of conventional CD4+ T, CD8+ T and NK cells were low. The Treg increase peaked on day 8 or 11 and lasted for over day 29. KKC80 showed a more sustained upregulation of functional Treg markers (e.g., Foxp3 and CD25) in comparison to Fc.IL-2 V91K. The half-life of KKC80 was calculated as 83.5 to 150 h. At high doses, inflammation-related adverse effects, including increased CRP (≥0.3 mg/kg) and deterioration of general conditions (1 mg/kg) were observed. In the mouse xenogenic GvHD model, KKC80 ameliorated GvHD symptoms and suppressed multiple tissue inflammation markers. Decreased soluble CD25 and IFN-γ were also confirmed, suggesting Treg-mediated anti-inflammatory effect by KKC80 administration were exerted in vivo. In the monkey DTH model, KKC80 suppressed skin inflammation and antibody production. Conclusion Among next-generation IL-2 variants, KKC80 showed a best-in-class biological profile for Treg activation. A drastic and sustained increase of Tregs with high Treg-selectivity and anti-inflammatory effects were observed in vivo . These data suggest that in comparison to current IL-2 therapy, KKC80 provides superior therapeutic index and efficacy in patients with autoimmune and inflammatory diseases. Figure 1. Disclosure of Interests Masahiro Ikeda Employee of: Kyowa Kirin Co., Ltd., Shinpei Yamaguchi Employee of: Kyowa Kirin Co., Ltd., Masumi Murakami Employee of: Kyowa Kirin Co., Ltd., Shigeki Takaoka Employee of: Kyowa Kirin Co., Ltd., Yasuko Sakaguchi Employee of: Kyowa Kirin Co., Ltd., Shunki Yasui Employee of: Kyowa Kirin Co., Ltd., Kousuke Iijima Employee of: Kyowa Kirin Co., Ltd., Kenichiro Nanya Employee of: Kyowa Kirin Co., Ltd., Hideyuki Onodera Employee of: Kyowa Kirin Co., Ltd., Toru Amano Employee of: Kyowa Kirin Co., Ltd.